The present invention relates to improved methods and compositions for the identification and quantitation of receptors on the surfaces of certain hematological cells. More particularly, the present invention relates to a simple rapid diagnostic method for the indication of bacterial infection, disease or immune disorder in mammals.
Pentraxins include, among other proteins, C-reactive protein (CRP), that was originally identified as a serum factor responsible for the precipitation of ‘acute phase’ patient sera with the somatic C polysaccharide (CPS) of pneumococcal cell walls. CRP has been shown to participate in reactions of precipitation, agglutination, opsonization and complement activation. These properties have been reproduced over the years in different laboratories. Conflicting reports of suppression, stimulation or chemoattraction of polymorphonuclear leukocytes (PMN) or monocytes, as well as the activation or inhibition of platelets by CRP have yet to be resolved.
Human CRP is a pentameric protein composed of identical 206 amino acid subunits (SEQ ID NO: 2), each having a molecular weight of 23,017 daltons that associate by non-covalent bonds (Mullenix and Mortensen, 1994 Mol. Immunol., 31(8):615-22). CRP can be dissociated to subunits by 8M urea or mild alkaline conditions only in the absence of calcium A single intramolecular disulfide bond links the two half-cysteines at Cys36 and Cys98. Human CRP is normally present in trace amounts in serum, e.g., 0.8-3 g/mL. However, during infection and inflammation, levels can increase a 1,000-fold in response to specific cytokines.
The single copy human CRP gene has been sequenced (Kilpatrick and Volanakis, 1991 Immunol. Res., 10(1):43-53). It contains two exons, one that encodes the signal peptide and the first two amino acids while the second contains the code for the remaining 204 amino acids and a long (1.2 kbp) 3′ untranslated region (UTR). No differences in coding regions have been found in libraries but the poly(GT) length of the intron exhibits polymorphic variation with three alleles containing 15, 19 or 22 repeats. CRP maps to chromosome 1 region q21 to q25.
CRP has been previously noted to bind to the cell walls of many bacteria, fungi and nematodes via the cell wall structural component phosphorylcholine (PC). In the presence of calcium, the primary ligand for CRP is PC. CRP binds its ligand, PC, at 1.9×10−5 M. Though PC is also a major component of mammalian cell membranes, CRP will bind to these membranes only under conditions that disturb the normal bilayer architecture. Many of the biological activities ascribed to CRP are initiated by binding ligands via the single PC-binding site within each subunit. Other reported ligands for CRP include small nuclear ribonucleoproteins (snRNP), fibronectin, lamnin, chromatin and histones.
The presence of CRP receptors (CRP-R) has been proposed for lymphocytes, NK cells, monocyte/macrophages and neutrophils. However, much of the prior art on CRP-R is conflicting. For example, reports of CRP-R on lymphocytes have implied an association with Fcγ receptors (FcγR) and a requirement for Ca2+ in conjunction with a CRP-PC complex. An increase in CRP-bearing lymphocytes during certain disease states has been reported (James, et al., 1982 Ann. NY. Acad. Sci., 389:274-85). From 1983 to 1991 various laboratories have reported the surface expression of CRP on lymphocytes along with de novo synthesis. Thus, the presence of a CRP-R on lymphocytes or surface expression of CRP has yet to be confirmed.
More recently, there have been reports of an inducible CRP receptor on PMA-stimulated neutrophils or a receptor for CRP on polymorphonuclear leukocytes (PMN) or neutrophils. It was reported that approximately 36% of resting PMN bound aggregated CRP compared to 93% when stimulated with PMA (Zeller, et al., 1986) and that 8% of the lymphocytes and 70% of the monocytes also bound aggregated CRP as detected by FITC-conjugated F(ab′)2 fragments of anti-CRP. Aggregated human IgG inhibited any binding by CRP leading to the suggested involvement of the Fc receptor. Still other reports indicated that both calcium and magnesium were necessary for binding to neutrophils. A CRP receptor on monocytes has been demonstrated in many laboratories under a variety of conditions. Approximately 40% of the peripheral blood monocytes and some mouse macrophage cell lines were reported to bind complexed CRP. The existence of a CRP-like determinant was reported on peripheral blood monocytes using polyclonal antibodies to CRP. Some papers have concluded that CRP-R is not FcγR, but may be associated with it.
Specific binding of radiolabeled CRP to isolated human peripheral blood monocytes (Ballou, et al., 1989 J. Immunol., 142(8):2708-13) was reported with a dissociation constant of about 10−7M, a requirement for calcium, an optimal pH of 7.4, and a lack of inhibition with human IgG. Other reports noted that an average of 67±12% of monocytes bound bCRP. Various publications in the 1990's have ascribed generation of H2O2 production, tumoricidal activity, induction of inflammatory cytokines, tissue factor and monocyte chemoattractant protein-1 to the internalization of the CRP receptor-bound ligand with subsequent degradation in human promonocyte U937 cells. Though there have been some reports of an increase in CRP-bearing lymphocytes during certain disease states, there have been no investigations of CRP binding to monocytes in any disease or its correlation to plasma CRP concentration.
Despite the considerable wealth of publications concerning CRP and its putative receptor(s), no biological role for C-reactive protein, the prototypic pentraxin, has been positively identified. The very nature and existence of the receptor is still under debate. Thus, there remains a need in the art for methods and compositions useful in the analysis of pentraxin-binding receptors in the presence of biological samples for the diagnosis of disease.